Mycobacterium tuberculosis, the causative agent of tuberculosis, is the leading cause of adult death by an infectious organism. Two outstanding characteristics of this organism are its complex cell envelope the biosynthesis of which is the target of several antibiotics, and its ability to persist in latently infected individuals. Major goals of the anti-mycobacterial drug development field include the identification of pathways that are essential for cell envelope biosynthesis and understanding the molecular mechanisms involved in the establishment of persistence. In this regard, one essential biosynthetic pathway that has been overlooked in this field is peptidoglycan (PGN) assembly. The PGN is an essential component of the cell envelope of virtually all bacteria, providing both shape and structural integrity to the cell. Mycobacterial PGN polysaccharide is composed of N-acylmuramic acid and N-acetylglucosamine with peptides (L-alanyl (or glycyl)-D-iso-glutaminyl-meso-diaminopimelyl-D-alanyl-D- alanine) attached to the muramic acid moieties. Peptide cross-links occur between meso-diaminopimelic acid (DAP) residues and either D-alanine or other DAP residues. The mycobacterial PGN is highly crosslinked with 70-80% of the peptides cross-linked and one-third to one-half of the cross-links represented by the DAP-DAP variety. In most other bacteria, DAP-DAP cross-links usually represent only a few percent of the total number of cross-links, suggesting an important role for these cross-links in mycobacterial physiology. The biosynthesis of these unusual cross-links and their function are unknown. It is thought that the enzymes responsible for DAP-DAP cross-links are insensitive to inhibition by R-lactam antibiotics and thus represent a novel class of enzymes to target for drug development. Furthermore, these linkages may be important for cell survival under long-term starvation conditions. However, the pathway for DAP-DAP linkage formation has not been described for any bacterial species. We propose that DAP-DAP linkages are essential for mycobacterial physiology and have a role in stationary phase survival. The formation of these cross-links may also be an important mechanism contributing to the establishment of persistence in tuberculosis latency. The overall goal of this proposal is to better understand cell wall assembly in mycobacteria with a focus on the biosynthesis and significance of DAP-DAP cross-links. Understanding the biology of these cross-links could lead to the development of new antibiotics for the management of latent tuberculosis, which would have a significant impact upon public health. Project Narrative The overall goal of this proposal is to better understand cell wall assembly in mycobacteria with a focus on the biosynthesis and significance of DAP-DAP cross-links. Understanding the biology of these cross-links could lead to the development of new antibiotics for the management of latent tuberculosis, which would have a significant impact upon public health.